Approximately 28,200 new cases of head and neck squamous cell carcinoma (HNSCC) will be diagnosed in 2004 with an estimated 7,200 deaths occurring. Clearly, additional local therapy is warranted to improve outcome. To determine the effectiveness of additional local treatment, a molecular imaging approach is proposed using a modified viral vector for both diagnostic and treatment purposes. This proposal will use molecular imaging to evaluate transgene expression in mice bearing HNSCC xenografts following administration of the viral vector. It is well established that hyperthermia enhances the cytotoxic effects of external beam ionizing radiation (IR). HNSCC has become a target of gene therapy and hyperthermic intervention strategies due to its anatomic accessibility. The enzyme herpes simplex virus type 1 thymidine kinase (HSV1-TK) in combination with ganciclovir (GCV) has been shown in vitro and in vivo to exert a cytotoxic effect after gene transfer of TK. A mutant version of TK (mTK) that enhances the cytotoxic effects of GCV and can be imaged by positron-emission tomography (PET) using positron-emitting nuclesoside analogs has been constructed. Therefore, improved imaging and therapy of HNSCC should be accomplished by gene transfer of mTK. However, one of the problems associated with gene transfer approaches has been the non-target expression of transgene. In this regard, we have constructed an adenovirus (AdHSmTK) encoding mTK under control of the inducible heat shock 70b (HS) promoter. This vector will only allow expression of mTK after exposure to heat. Using ultrasound technology we can deliver focused heat to xenografts that parallels clinical delivery. The specific aims are: 1.) Characterize the time course and level of expression of mTK in HNSCC cells after infection with AdHSmTK and determine the cytotoxicity after exposure to GCV and IR; 2.) Characterize the time course and level of expression of mTK in nude mice bearing HNSCC xenografts after infection with AdHSmTK with microPET imaging following exposure to heat. We hypothesize that the combination of gene therapy with hyperthermia and IR will have a greater impact than either alone or combination of two of the modalities in vitro and that the use of molecular imaging to optimize the system in vivo will lead to a rational treatment protocol combining these modalities in the future.